CN117327739B - MiR-503-322在构建急性和慢性胰腺炎动物模型中的应用 - Google Patents
MiR-503-322在构建急性和慢性胰腺炎动物模型中的应用 Download PDFInfo
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Abstract
本发明公开了miR‑503‑322在构建急性和慢性胰腺炎动物模型中的应用,属于动物模型构建技术领域。本发明通过基因编辑的方法构建了一种成年诱导型胰腺腺泡细胞特异性过表达miR‑503‑322的基因敲入小鼠模型(KI‑miR‑503‑322Ela+小鼠),该小鼠模型由于腺泡细胞中miR‑503‑322的诱导性高表达,自发引起腺泡细胞胰酶激活,导致腺泡细胞坏死和胰腺炎的急性发作;在急性胰腺炎发作后,该模型小鼠还可抑制腺泡细胞增殖,进而促发了慢性胰腺炎。本发明的模型可用于研究急、慢性胰腺炎的发病机理,并进一步筛选预防和/或治疗胰腺炎的药物。
Description
技术领域
本发明属于动物模型构建技术领域,具体涉及一种利用miR-503-322构建急性和慢性胰腺炎动物模型的方法。
背景技术
急性胰腺炎(Acute pancreatitis,AP)是最常见的胃肠道疾病住院治疗的原因之一,是多种病因导致胰腺组织自身消化所致的胰腺水肿、出血及坏死等炎性损伤,病情变化复杂。AP急性起病,常伴有上腹剧痛和血清胰酶升高,重症患者炎症波及全身,病情凶险,病死率高,预后差。AP患者容易反复发作,进展为慢性胰腺炎(Chronic pancreatitis,CP),主要特征为持续性炎症、进行性纤维化,最终导致胰腺内外分泌功能不可逆损伤,致使患者长期健康生活质量显著下降。随着急、慢性胰腺炎发病率的逐年攀升,对其发病机理的探索和治疗药物的开发显得尤为重要,而临床研究尚存在伦理争议,可以模拟胰腺炎发病过程的动物模型则是更好的研究工具。因此,建立急、慢性胰腺炎动物模型来深入探索胰腺炎的发病机理,对筛选预防和/或治疗胰腺炎的药物具有重要意义。
动物模型,尤其是小鼠动物模型,是研究人类发病机制、筛选药物治疗靶点和开发治疗手段的主要工具。其中,基因改造动物模型已出现在各个疾病领域,具有非常广阔的研究以及应用前景。而目前胰腺炎动物模型主要采取的方法是通过外源性物理、化学、生物以及复合的致病因素作用于动物,造成动物胰腺局部损伤或伴有全身其他器官功能的损伤,部分模拟人类胰腺炎发病时的特点。例如,雨蛙素腹腔注射是最常见的诱导AP和CP的方式,虽简单易重复,但因其是化学药物与临床并无相关性;其次,胰胆管结扎或逆行注射胆汁酸也较为常见,但操作复杂,不易重复。其他模型基本无临床相关性,故目前研究使用较少,也难以为实际临床所面临的对胰腺炎的治疗手段提供较好的参考价值。
总之,在胰腺炎研究领域,缺乏相应的体内基因改造动物模型,所以本领域亟需解决如何获得一种更符合疾病病理特征,可为研究胰腺炎的发病机制和筛选药物提供疾病动物模型的技术问题。
微RNA(miRNAs)是一类长度约22个核苷酸的非编码RNA,在细胞核内由基因序列转录成初级转录产物(Pri-miRNAs),代表细胞内的原始转录水平,经过多次剪切后成为成熟的miRNAs。通过结合到靶基因mRNA的3’非翻译区(3’-UTRs)抑制蛋白质翻译或降解mRNA发挥转录后调控作用。已有多种研究报导了miRNAs在胰腺炎中发挥重要作用。比如,miR-135a和miR-22在AP动物模型的胰腺组织中明显上调,促进腺泡细胞凋亡;miR-19b可诱导腺泡坏死,在重症胰腺炎中表达显著增加。
MiR-503-322簇位于X染色体长臂26.3位点,包括miR-503和miR-322,它们具有相同的种子序列(CGACGA)。研究发现胚胎期胰腺中miR-503高表达与胰腺发育相关,出生后表达逐渐降低,而miR-503-322在病理条件下重新开放表达能够促进多种疾病的发生,如糖尿病坏疽等。
发明内容
本发明的目的在于提供miR-503-322在构建急性和慢性胰腺炎动物模型中的应用。
具体地,所述动物模型的腺泡细胞特异性过表达miR-503-322基因,所述miR-503-322基因的核苷酸序列如SEQ ID NO.1所示。
进一步地,所述腺泡细胞为胰腺外分泌腺腺泡细胞;所述动物为哺乳动物。优选地,所述动物为小鼠。
进一步优选地,所述动物为C57BL/6J小鼠。
进一步地,所述方法包括以下步骤:
(1)基于CRISPR-Cas9系统设计靶向小鼠miR-503-322基因的sgRNA,其序列如SEQID NO.2所示;
(2)合成小鼠CAG-LSL-miR-503-322片段,其核苷酸序列如SEQ ID NO.3所示,基于CRISPR-Cas9系统将小鼠CAG-LSL-miR-503-322片段重组到靶位点H11上,构建miR-503-322条件性敲入动物模型,命名为miR-503-322 KI小鼠;其中,CAG-LSL-miR-503-322片段可由公司合成。靶位点即H11位点,CRISPR-Cas9常用的位点;
(3)将miR-503-322 KI小鼠与Elastase-CreER工具小鼠交配,通过小鼠基因型鉴定筛选得到Elastase-Cre阳性并且miR-503-322 KI杂合的后代,得到KI-miR-503-322Ela+杂合小鼠,即诱导型腺泡细胞特异性miR-503-322基因敲入小鼠。
具体地,用于小鼠基因型鉴定的特异性引物对包括KI-1引物对、KI-4引物对、WT-1引物对和WT-2引物对,其中,
KI-1引物对为:
KI-1-F:5’-TGTCTGGATCCCCATCAAGCTG-3’;
KI-1-R:5’-GATCTGCTGGTGAGGCAAAGGGT-3’;
KI-4引物对为:
KI-4-F:5’-TTCAGCTGCCCACTCTACTG-3’;
KI-4-R:5’-GACCTCTGAAAGACCAGCTA-3’;
WT-1引物对为:
WT-1-F:5’-CCTTAAAGTTGTACGGAAGAGTCGGGT-3’;
WT-1-R:5’-CCACCTTTGCCCCAGATACCAAGAC3’;
WT-2引物对为:
WT-2-F:5’-CTCTATGACCTGCTGCTGGAGGCG-3’;
WT-2-R:5’-CCACCTTTGCCCCAGATACCAAGAC-3’。
本发明进一步提出了一种急性或慢性胰腺炎动物模型的构建方法,通过将构建得到的miR-503-322基因敲入小鼠腹腔注射他莫昔芬溶液,获得腺泡细胞特异性过表达miR-503-322的基因敲入小鼠模型。
其中,中腔注射他莫昔芬的时机为小鼠成年期,注射剂量为100 mg/kg/天,连续注射2天。AP模型在首次他莫昔芬注射后2天得到,CP模型在首次注射他莫昔芬后28天得到。成年期即8-12周龄,即小鼠性成熟以后。
所述他莫昔芬溶液通过将75~100 mg他莫昔芬粉末溶解于5 mL玉米油中得到。
通过上述方法构建得到的急、慢性胰腺炎疾病动物模型也在本发明的保护范围内。
本发明还提供了上述方法构建得到的急、慢性胰腺炎动物模型在筛选预防和/或治疗胰腺炎的药物中的应用。
本发明通过基因修饰的方法成功构建了一种可诱导的胰腺腺泡细胞特异性过表达miR-503-322的基因敲入小鼠模型,该小鼠模型由于腺泡细胞中miR-503-322的高表达,导致小鼠胰腺腺泡细胞胰酶分泌异常和过早激活,诱发了AP;该小鼠模型腺泡细胞增殖抑制,损伤了胰腺自我修复,进而在一月内就形成了CP。该模型可以用于进一步研究急、慢性胰腺炎的发病机理,筛选预防和/或治疗胰腺炎的药物。
与现有的小鼠胰腺炎模型相比,本发明采用先进的基因编辑技术,在遗传物质DNA上进行精准修饰,从而获得了可稳定遗传的小鼠动物模型。该模型无需雨蛙素等化学刺激或者胰胆管结扎等物理刺激,即可自发急、慢性胰腺炎,并且类似人类胰腺炎的发病机理,继而用于疾病的致病机制和治疗研究前景广阔。
附图说明
图1显示的是诱导型腺泡细胞特异性miR-503-322高表达模型小鼠的构建,其中A显示的是构建方案,对照小鼠为KI-miR-503-322Ela-,模型小鼠为KI-miR-503-322Ela+;B显示的是定量PCR检测腺泡细胞Pri-miR-503-322的表达水平。与对照相比,**P<0.01。
图2显示的是对照和模型小鼠诱导后2天血清淀粉酶和脂肪酶水平变化,其中A显示的是淀粉酶水平;B显示的是脂肪酶水平。与对照相比,*P<0.05,***P<0.001。
图3显示的是对照和模型小鼠诱导后2天胰腺组织病理变化,其中A和B显示的是胰腺H&E;C显示的胰腺巨噬细胞免疫荧光染色;D显示的是600倍镜下巨噬细胞数量统计;E显示的是组织损伤评分。与对照相比,***P<0.001。
图4显示的是对照和模型小鼠诱导后28天胰腺组织病理变化。其中A显示的局部放大区域是纤维化;B显示的局部放大区域是脂肪替代;C显示的局部放大区域是腺泡-导管化生。
图5显示的是KI-miR-503-322Ela+小鼠腺泡细胞酶原激活。其中A显示的胰蛋酶原激活(绿色荧光);B显示的是血清胰蛋白酶活性检测。与对照相比,**P<0.01。
图6显示的是KI-miR-503-322Ela+小鼠腺泡细胞增殖抑制。其中A显示的增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)(绿色)、淀粉酶(红色)和细胞核(蓝色)免疫荧光染色,箭头代表增殖的腺泡细胞,星号代表增殖的间质细胞;B显示的是600倍镜下增殖的腺泡细胞数目统计。与对照相比,**P<0.01。
实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
本发明所用原料与设备均为已知产品,通过购买市售产品所得。
本发明所有动物实验均获得南京医科大学实验动物管理委员会的许可(伦理号:IACUC-2004040),所有实验动物均饲养于南京医科大学实验动物中心屏障设施内,在清洁环境中饲养,温度(21±2)℃,湿度(35±2)%,12 h昼夜间断照明,自由进食饮水,饮用水为实验动物中心制备的无菌水。
实施例1 构建诱导型腺泡细胞特异性miR-503-322高表达模型小鼠。
实验方法:
(1) 基于CRISPR-Cas9系统设计靶向小鼠miR-503-322基因(SEQ ID NO.1)的sgRNA,其序列为SEQ ID NO.2:5’-CTGAGCCAACAGTGGTAGTA -3’;
(2) 基于CRISPR-Cas9系统将小鼠CAG-LSL-miR-503-322片段(片段序列如SEQ IDNO.3所示)重组到靶位点H11位点上,成功构建miR-503-322 KI小鼠;
(3) 将miR-503-322 KI小鼠与Elastase-CreER(由四川大学华西医学院赠送)工具小鼠交配,后代通过小鼠基因型鉴定筛选得到Elastase-Cre阳性并且miR-503-322 KI杂合的后代,即基因型为KI-miR-503-322Ela+的阳性小鼠;
(4) 模型小鼠配繁获得的F1代基因型鉴定PCR结果判读方法如下:KI-1和KI-4引物用于区分miR-503-322野生型和敲入型;WT-1,WT-2引物用于区分Elastase-CreER阳性和阴性。PCR产物凝胶电泳具体条带为:KI-1引物野生型条带为0 bp,敲入型为425 bp;KI-4引物野生型条带为420 bp,敲入型为0 bp;WT-1引物野生型为500 bp,突变型为3000 bp;WT-2引物野生型条带为0 bp,突变型为394 bp。KI-1,KI-4,WT-1,WT-2引物PCR产物同时出现425bp、420 bp、3000 bp和394 bp即为阳性模型小鼠即KI-miR-503-322Ela+;KI-1,KI-4,WT-1,WT-2引物PCR产物同时出现425 bp、420 bp、500 bp和0 bp,则为阴性对照小鼠即KI-miR-503-322Ela-。用于小鼠基因型鉴定的特异性引物如下:
KI-1引物对为:
KI-1-F:5’-TGTCTGGATCCCCATCAAGCTG-3’;
KI-1-R:5’-GATCTGCTGGTGAGGCAAAGGGT-3’;
KI-4引物对为:
KI-4-F:5’-TTCAGCTGCCCACTCTACTG-3’;
KI-4-R:5’-GACCTCTGAAAGACCAGCTA-3’;
WT-1引物对为:
WT-1-F:5’-CCTTAAAGTTGTACGGAAGAGTCGGGT-3’;
WT-1-R:5’-CCACCTTTGCCCCAGATACCAAGAC3’;
WT-2引物对为:
WT-2-F:5’-CTCTATGACCTGCTGCTGGAGGCG-3’;
WT-2-R:5’-CCACCTTTGCCCCAGATACCAAGAC-3’。
(5) 他莫昔芬工作液配制:100 mg他莫昔芬粉末(Sigma,America)溶解在5 mL玉米油(Sigma,America),充分溶解混匀,备用。
(6) 对Elastase-CreER-miR-503-322 KI小鼠腹腔注射他莫昔芬连续2天,注射剂量为100 mg/kg/天,获得腺泡细胞特异性过表达miR-503-322的基因敲入小鼠模型。
(7) 分离对照小鼠腺泡细胞和模型小鼠腺泡细胞,定量PCR检测Pri-miR-503(初级转录本)的表达水平。
实验结果:如图1所示,他莫昔芬腹腔注射2天后,相比于对照小鼠,模型小鼠腺泡细胞Pri-miR-503的表达水平明显升高。这一结果表明,本发明成功构建了一个可诱导的腺泡细胞特异性高表达miR-503-322的基因敲入小鼠模型。
下面结合试验对本发明的技术效果作详细的描述。
如图2和图3所示,他莫昔芬诱导2天后,模型小鼠血清淀粉酶、脂肪酶的水平和胰腺组织病理学变化可以明确模型小鼠的AP表型。
循环淀粉酶、脂肪酶水平升高是AP的重要标志,也是作为临床诊断的标准之一。采用商品化试剂盒(南京建成生物工程研究所,中国)检测血清淀粉酶活力;采用酶联免疫吸附技术检测试剂盒(Elabscience,中国)检测脂肪酶水平。结果如图2中的A和B所示,他莫昔芬诱导2天后,相比对照小鼠,模型小鼠血清淀粉酶和脂肪酶水平明显升高,说明该模型小鼠能够成功构建具有显著AP特征的动物模型。
病理诊断是疾病的金标准。为进一步观察小鼠胰腺组织的病理改变,将胰腺组织经石蜡包埋切片后进行H&E染色(武汉塞维尔生物,中国)。结果如图3中的A和B显示,他莫昔芬诱导2天后,对照小鼠胰腺组织腺泡细胞完整,排列规则,无间质水肿和炎细胞浸润,说明他莫昔芬本身对胰腺组织并无影响。相比之下,模型小鼠胰腺组织间隙明显增加,腺泡细胞水肿分离,导管和小叶间可见明显炎细胞浸润,局部可见腺泡细胞坏死。巨噬细胞是AP过程中常见的炎细胞,如图3中的C和D显示,模型小鼠巨噬细胞数目显著增加。对小鼠胰腺切片从水肿、出血、炎细胞浸润和坏死四个方面进行评分,结果如图3中的E显示,模型小鼠组织损伤评分明显增高。
以上结果表明,腺泡细胞特异性高表达miR-503-322的小鼠经他莫昔芬诱导后可以导致小鼠血清淀粉酶、脂肪酶水平升高以及胰腺病理损伤,标志着AP的发生,通过本发明的构建方法,能够有效构建特征明显的AP模型。
实施例2 利用腺泡细胞高表达miR-503-322模型小鼠构建CP模型。
实验方法:将模型小鼠KI-miR-503-322Ela+和对照小鼠KI-miR-503-322Ela-按照实施例1步骤分别连续腹腔注射他莫昔芬两天,连续监测28天,分别取小鼠胰腺组织进行病理检测。
实验结果:病理学诊断是CP的确定标准。如图4所示,模型小鼠经他莫昔芬诱导后的第28天,胰腺切片经马松染色后可见明显胶原沉积,说明纤维化明显。脂肪替代和腺泡-导管化生也明显存在,这些都是明显的CP特征性形态学改变。
以上结果表明,腺泡细胞特异性高表达miR-503-322的小鼠经他莫昔芬诱导后28天,导致典型的CP病理学改变,标志着CP的发生,通过本发明的构建方法,能够有效构建特征明显的CP模型。
实施例3 KI-miR-503-322Ela+小鼠腺泡细胞酶原激活。
实验方法:胰腺腺泡细胞内酶原激活是胰腺炎发生的主要病理机制,罗丹明可以用于检测激活的胰蛋白酶。因此,我们分离对照小鼠和模型小鼠的腺泡细胞,将新鲜提取的腺泡细胞均匀种在共聚焦皿内,加入终浓度10 μmol的胰蛋白酶底物罗丹明(Thermo,美国),37℃孵育30 min后,用激光共聚焦显微镜在激发光488 nm处捕捉荧光。
实验结果:如图5中的A所示,模型小鼠腺泡内可见明显的绿色荧光即激活的胰蛋白酶。如图5中的B所示,模型小鼠血清中胰蛋白酶活性也明显增强。这一结果说明,miR-503-322的高表达导致胰蛋白酶原激活,这一胰腺炎模型小鼠可模拟人类胰腺炎发病机制。
实施例4KI-miR-503-322Ela+小鼠腺泡细胞增殖抑制。
实验方法:AP发生后,会刺激腺泡细胞的增殖来进行自我修复。如果腺泡细胞增殖能力不足,会加速AP向CP发展。因此,我们利用PCNA的免疫荧光染色检测了模型小鼠的腺泡细胞增殖能力。
PCNA免疫荧光染色方法:胰腺组织的石蜡切片经脱蜡、梯度酒精水化后,用碱性修复液进行抗原修复。待自然冷却后,PBS冲洗,用3% BSA室温封闭30 min,随后用一抗即PCNA(Abcam,美国,1:100稀释)4℃孵育过夜。然后PBS清洗后用荧光二抗Alexa FluorTM-488(Proteintech,美国,1:500稀释)37℃ 孵育1 h。PBS清洗后继续加入第二种一抗即淀粉酶(Santa,美国,1:100稀释)37℃ 孵育2 h后,PBS清洗以后加入荧光二抗Alexa FluorTM-594(Proteintech,美国,1:500稀释)37℃ 孵育1 h。PBS清洗后用Hoechst 33342(碧云天,中国,1:8000稀释)染细胞核,室温8 min。最后用PBS清洗后滴加防淬灭剂(Invitrogen,美国)10 μL,用盖玻片封片。完成后在激光共聚焦显微镜下观察。
实验结果:如图6所示,与淀粉酶共染阳性的细胞即为腺泡细胞,可见模型小鼠胰腺中增殖的腺泡细胞明显减少,间质细胞增殖增多。表明miR-503-322的过表达抑制腺泡细胞的增殖,损伤胰腺修复,促进CP的进展。
总体来看,本发明按照实施例1和2的方法构建的急性和慢性胰腺炎模型,模型小鼠在F5代依然具有明显的AP和CP特征,说明本发明方法遗传性好,成功率高。
综上,本发明提供了miR-503-322在构建急性和慢性胰腺炎动物模型中的应用。本发明通过基因编辑的方法成功构建了一种成年期诱导型的胰腺腺泡细胞特异性过表达miR-503-322的基因敲入模型小鼠(KI-miR-503-322Ela+)。该模型小鼠由于腺泡细胞内miR-503-322的诱导性高表达,导致腺泡细胞酶原激活和增殖抑制,诱发急性和慢性胰腺炎。该模型可用于研究胰腺炎的发病机理,并进一步筛选预防和/或治疗胰腺炎的药物,具有深远的临床意义和推广价值。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其他多种形式的修改、替换或变更。以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
Claims (10)
1.MiR-503-322基因在构建急性或慢性胰腺炎动物模型中的应用,其中,所述动物模型的腺泡细胞特异性过表达miR-503-322基因,所述miR-503-322基因的核苷酸序列如SEQ IDNO.1所示。
2.根据权利要求1所述的应用,其特征在于,所述腺泡细胞为胰腺外分泌腺腺泡细胞;所述动物为哺乳动物。
3.根据权利要求1所述的应用,其特征在于,所述动物为C57BL/6J小鼠。
4.一种miR-503-322基因敲入小鼠模型的构建方法,其特征在于,包括如下步骤:
(1)基于CRISPR-Cas9系统设计靶向小鼠miR-503-322基因的sgRNA,其序列如SEQ IDNO.2所示;
(2)合成小鼠CAG-LSL-miR-503-322片段,其核苷酸序列如SEQ ID NO.3所示,基于CRISPR-Cas9系统将小鼠CAG-LSL-miR-503-322片段重组到靶位点H11上,构建miR-503-322条件性敲入动物模型,命名为miR-503-322 KI小鼠;
(3)将miR-503-322 KI小鼠与Elastase-CreER工具小鼠交配,通过小鼠基因型鉴定筛选得到Elastase-Cre阳性并且miR-503-322 KI杂合的后代,命名为KI-miR-503-322Ela+杂合小鼠,即得到诱导型腺泡细胞特异性miR-503-322基因敲入小鼠。
5.根据权利要求4所述的构建方法,其特征在于,用于小鼠基因型鉴定的特异性引物对包括KI-1引物对、KI-4引物对、WT-1引物对和WT-2引物对,其中,
KI-1引物对为:
KI-1-F:5’-TGTCTGGATCCCCATCAAGCTG-3’;
KI-1-R:5’-GATCTGCTGGTGAGGCAAAGGGT-3’;
KI-4引物对为:
KI-4-F:5’-TTCAGCTGCCCACTCTACTG-3’;
KI-4-R:5’-GACCTCTGAAAGACCAGCTA-3’;
WT-1引物对为:
WT-1-F:5’-CCTTAAAGTTGTACGGAAGAGTCGGGT-3’;
WT-1-R:5’-CCACCTTTGCCCCAGATACCAAGAC3’;
WT-2引物对为:
WT-2-F:5’-CTCTATGACCTGCTGCTGGAGGCG-3’;
WT-2-R:5’-CCACCTTTGCCCCAGATACCAAGAC-3’。
6.一种急性或慢性胰腺炎动物模型的构建方法,其特征在于,向权利要求4或5构建得到的诱导型腺泡细胞miR-503-322基因敲入小鼠腹腔注射他莫昔芬溶液,获得腺泡细胞特异性过表达miR-503-322的基因敲入小鼠模型。
7.根据权利要求6所述的构建方法,其特征在于,腹腔注射他莫昔芬溶液的时机为小鼠成年期,注射剂量为100 mg/kg/天,连续注射2天;急性胰腺炎动物模型在首次他莫昔芬溶液注射2天后即可得到,慢性胰腺炎动物模型在首次注射他莫昔芬溶液28天后得到。
8.根据权利要求6或7所述的构建方法,其特征在于,所述他莫昔芬溶液通过将75~100mg他莫昔芬粉末溶解于5mL玉米油中得到。
9.权利要求6~7任一项所述的构建方法得到的急性或慢性胰腺炎动物模型。
10.权利要求9构建得到的急性或慢性胰腺炎动物模型在筛选预防和/或治疗胰腺炎的药物中的应用。
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